Induction of a humoral response is important in host defense, for example, in fighting infections by pathogens. A humoral response is mediated by B cells, but requires the help of other cells, such as T cells. Development of a humoral response is a multistage process which occurs primarily in secondary lymphoid tissues.
Resting B cells circulate in the blood, pass through secondary lymphoid tissues, such as lymph nodes, Peyer""s patches, spleen and tonsils, where they come into contact with trapped antigens. B cells recognizing a specific antigen through their surface immunoglobulins process the antigen and enter the T cell rich paracortical regions below the outer layer, or cortex, of lymphoid tissues. Some of the T cells in this region have been activated through contact with antigen presenting cells. These T cells in turn promote B cell activation by direct contact with the B cells via interaction of cell surface molecules and by the secretion of T cell-specific cytokines, such as IL-2, IL-4, and IL-5. This cell-cell interaction between T and B cells or production of T cell specific cytokines induces B cells to migrate into B cell follicles. In these follicles, the B cells interact with follicular dendritic cells having antigen-antibody complexes on their surface resulting in the formation of a germinal center. B cells in germinal centers undergo active proliferation, affinity maturation, and differentiation into memory B cells or antibody secreting plasma cells. Thus, whereas antibody-secreting plasma cells produce antibodies to fight infections, memory B cells assure a more rapid response to subsequent exposure to the same antigen.
Some molecules involved in the induction of B-cell proliferation and differentiation have been identified. In addition to cytokines produced in large part by activated T cells, crosslinking of specific B cell surface molecules also provide such signals (Clark E. A. et al, (1994), Nature 367, 425). One such B cell surface molecule is CD40. CD40 is a 45-50 kD protein expressed on activated B cells. Valle et al., (1989), Eur. J Immunol, 19:1463-1467; Gordon et al., (1988), J Immunol., 140:1425-1430; Gruber et al., (1989), J Immunol, 142: 4144-4152. Crosslinking of CD40 with antibodies or with its natural ligand, CD40L, also termed gp39, together with other stimulatory signals induces B cell proliferation and antibody production. Armitage et al., (1992), Nature, 357:80-82 ; Hollenbaugh et al., (1992), EMBO J, 11:4313-4319.
T cells are not only required for providing help to B cells, but also play a major role in cellular immune responses, such as in delayed type hypersensitivity reactions and in cytotoxicity. To exert their activity, T cells must be activated. To induce antigen-specific T cell activation and clonal expansion, two signals provided by antigen-presenting cells (APCs) must be delivered to the surface of resting T lymphocytes (Jenkins, M. and Schwartz, R. (1987) J. Exp. Med 165, 302-319; Mueller, D. L., et al. (1990) J Immunol. 144, 3701-3709; Williams, I. R. and Unanue, E. R. (1990) J Immunol. 145, 85-93). The first signal, which confers specificity to the immune response, is mediated via the T cell receptor (TCR) following recognition of foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second signal, termed costimulation, induces T cells to proliferate and become functional (Schwartz, R. H. (1990) Science 248, 1349-1356). Costimulation is neither antigen-specific, nor MHC restricted and is thought to be provided by one or more distinct cell surface molecules expressed by APCs (Jenkins, M. K., et al. (1988) J Immunol. 140, 3324-3330; Linsley, P. S., et al. (1991) J. Exp. Med 173, 721-730; Gimmi, C. D., et al., (1991) Proc. Natl. Acad Sci. USA. 88, 6575-6579; Young, J. W., et al. (1 992) J Clin. Invest. 90, 229-237; Koulova, L., et al. (I 991) J. Exp. Med 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89, 271-275; van-Seventer, G. A., et al. (1990) J. Immunol. 144, 4579-4586; LaSalle, J. M., et al., (I 991) J Immunol. 147, 774-80; Dustin, M. I., et al., (1989) J Exp. Med 169, 503; Armitage, R. J., et al. (1992) Nature 357, 80-82; Liu, Y., et al. (1992) J. Exp. Med. 175, 437-445). B7- 1 and B7-2 are two such costimulatory molecules which interact with CD28 and CTLA4 on T cells (Linsley, P. S., et al., (1991) J. Exp. Med. 173, 721-730; Gimmi, C. D., et al., (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Koulova, L., et al., (1991) J Exp. Med. 173, 759-762; Reiser, H., et al. (1992) Proc. Natl. Acad. Sci. USA. 89, 271-275; Linsley, P. S. et al. (1990) Proc. Natl. Acad. Sci. USA. 87, 5031-5035; Freeman, G. J. et al. (1991) J Exp. Med 174,625-631; Freeman, G. J. et al. (1993) Science 262:909-911; Azuma, M. et al. (1993) Nature 366:76-79; and Freeman, G. J. et al. (993) J Exp. Med 178:2185-2192). Though the molecules B7-1 and B7-2 play a critical role in costimulation of T cell, there is some evidence that additional molecules can provide a costimulatory singnal to T cells.
Previous studies demonstrated that several antibodies recognized a 150kD cell-surface homodimer, termed CD100, that is expressed on a number of hematopoietic cells including B and T lymphocytes, granulocytes, monocytes and natural killer cells but not on eosinophils, platelets, erythrocytes or hematopoietic progenitor cells (Bougeret, C. et al., (1992), J Immunol. 148, 318; Herold, C., et al., Eds., (Oxford University Press, Oxford, 1995), Leucocyte Typing V., S. F. Schlossman, et al., Eds. vol. 1, pp. 52). These studies indicated that CD100 expression on resting T cells increases on T cells after phytohemagglutinin activation (Bougeret, C. et al., (1992), J Immunol. 148, 318). In addition, it has been reported that crosslinking of CD 100 provides a costimulatory signal to T cells, indicating that this molecule may be involved in T cell activation and clonal expansion (Herold C. et al., (1994), Int. Immunol. 7, 1). However, the role of CD 100, in particular its role on B lymphocytes is unknown.
This invention provides isolated nucleic acid molecules encoding a CD100 antigen. Such nucleic acid molecules (e.g., cDNAs) have a nucleotide sequence encoding a CD100 antigen or biolocially active portions thereof, such as a peptide having a CD100 activity. In a preferred embodiment, the isolated nucleic acid molecule has a nucleotide sequence shown in FIG. 1, SEQ ID NO: 1, or a portion thereof such as the coding region of the nucleotide sequence of FIG. 1, SEQ ID NO: 1. Other preferred nucleic acid molecules encode a protein having the amino acid sequence of FIG. 2, SEQ ID NO: 2. Nucleic acid molecules derived from hematopoietic cells (e.g., a naturally-occurring nucleic acid molecule found in an activated lymphocyte) which hybridize under stringent conditions to the nucleotide sequence shown in FIG. 1, SEQ ID NO: 1 are also within the scope of the invention.
In another embodiment, the isolated nucleic acid molecule is a nucleotide sequence encoding a protein having an amino acid sequence which is at least about 80%, preferably at least about 85%, more preferably at least about 90% and most preferably at least about 95-99% overall amino acid sequence identity with an amino acid sequence shown in FIG. 2, SEQ ID NO: 2. This invention further pertains to nucleic acid molecules which encode a protein which includes a semaphorin domain having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2. Also within the scope of this invention are nucleic acid molecules which encode a protein which includes an extracellular domain having an amino acid sequence at least 80%, preferably at least 90%, more preferably at least 95-99% identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2.
Nucleic acid molecules encoding proteins which include a semaphorin domain having an amino acid sequence at least 60% (preferably at least 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2 and an immunoglobulin-like domain having an amino acid sequence at least 50% (preferably at least 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2 are also within the scope of this invention. These nucleic acid molecules can encode proteins which optionally include a cytoplasmic domain having an amino acid sequence at least 50% (preferably at least 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2.
Another aspect of this invention pertains to nucleic acid molecules encoding a CD100 fusion protein which includes a nucleotide sequence encoding a first peptide having an amino acid sequence at least 80% (preferably at least 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2, SEQ ID NO: 2 and a nucleic sequence encoding a second peptide corresponding to a moiety that facilitates detection or purification of the molecules or that alter the solubility, binding affinity or valency of the first peptide, such as an immunoglobulin constant region.
In another embodiment, the isolated nucleic acid molecule is a nucleotide sequence encoding a peptide fragment of at least about 30 amino acid residues in length, preferably at least about 40 amino acid residues in length, and more preferably at least about 50 amino acid residues in length corresponding to the amino acid sequence shown in FIG. 2, SEQ ID NO: 2. In a preferred embodiment, the peptide fragment has a CD100 activity.
Moreover, given the disclosure herein of a CD100-encoding cDNA sequence (e.g., SEQ ID NO: 1), antisense nucleic acid molecules (i.e, molecules which are complimentary to the coding strand of the CD100 cDNA sequence) are also provided by the invention.
Another aspect of the invention pertains to recombinant expression vectors containing the nucleic acid molecules of the invention and host cells into which such recombinant expression vectors have been introduced. In one embodiment, such a host cell is used to produce CD100 protein by culturing the host cell in a suitable medium. If desired, CD100 protein can be then isolated from the medium or the host cell.
Still another aspect of the invention pertains to isolated CD100 proteins and active fragments thereof, such as peptides having an activity of a CD100 antigen (e.g., at least one biological activity of CD100, such as the ability to stimulate a B cell response, for example, B cell aggregation or the ability to stimulate a T cell response, for example, T cell proliferation). The invention also provides an isolated preparation of a CD100 protein. In preferred embodiments, the CD100 protein comprises an amino acid sequence of FIG. 2, SEQ ID NO: 2, or a mature CD100 protein lacking an amino-terminal signal sequence (e.g., amino acids 22-863 of FIG. 2, SEQ ID NO: 2). In other embodiments, the isolated CD100 protein comprises an amino acid sequence at least 80 % identical to an amino acid sequence of FIG. 2, SEQ ID NO: 2 and, preferably has an activity of CD100 (e.g., at least one biological activity of CD100). Preferably, the protein is at least about 90 %, more preferably at least about 95 %, even more preferably at least about 98 % and most preferably at least about 99 % identical to the amino acid sequence of FIG. 2, SEQ ID NO: 2.
This invention also pertains to isolated peptides which include a semaphorin domain having an amino acid sequence that is at least 60% (preferably at least 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and an immunoglobulin-like domain having an amino acid sequence that is at least 50% (preferably at least 60%, 70%, 80%, 90%, or 95-99%) identical to an amino acid sequence shown in FIG. 2 (SEQ ID NO: 2). These peptides can optionally include a cytoplasmic domain having an amino acid sequence that is at least 50% identical to an amino acid sequence shown in FIG. 2 (SEQ ID NO: 2).
The invention also provides for a CD100 fusion protein comprising a first peptide having an amino acid sequence at least 80% identical to an amino acid sequence shown in FIG. 2 (SEQ ID NO: 2) and a second peptide corresponding to a moiety that alters the solubility, binding affinity or valency of the first peptide. In preferred embodiments, the fusion protein comprises an extracellular domain of a CD100 antigen. In yet another embodiment, the fusion protein comprises a semaphorin domain of a CD100 antigen.
Peptides comprising a fragment of at least about 30 amino acids, a fragment of at least about 40 amino acids, a fragment of at least about 50 amino acids, or longer fragments of the sequence shown in FIG. 2 (SEQ ID NO: 2) are also within the scope of the invention. The peptide fragments preferably have a CD100 activity.
A CD100 protein of the invention can be incorporated into a pharmaceutical composition which includes the protein (or active portion thereof) and a pharmaceutically acceptable carrier. In addition, vaccine compositions which include at least one antigen and a first agent which stimulates a CD100 ligand-associated signal in a cell, such as a leukocyte, e.g., a B cell or a T cell are also within the scope of this invention. Such vaccine compositions can further include a second agent which stimulates a CD40-associated signal in a cell (e.g., a B cell).
The CD100 protein of the invention and agents which modulate a CD100 ligand-associated signal in a cell, such as a leukocyte can be used to modulate leukocyte responses in vitro or in vivo. In one embodiment, the invention provides a method for stimulating a CD100 ligand-associated signal in a B cell by contacting the cell with an agent that stimulates a CD100 ligand-associated signal. Such an agent can be, for example, a stimulatory form of a CD100 antigen (e.g., a soluble CD100 protein). Additional agents, such as an agent which provides a CD40 associated signal in the B cell can be used to stimulate a B cell response. In another embodiment, a CD100 ligand-associated signal is inhibited to thereby inhibit a response by a B cell. In this embodiment, an agent which interacts with CD100, such as an anti-CD100 antibody can be used to inhibit a response by a B cell such as aggregation or differentiation. The methods of the invention for modulating B cell responses by manipulating the interaction of CD100 with its ligand can be applied in vitro (e.g., with cells in culture) or in vivo, wherein an agent that modulates CD100 and/or CD100 ligand is administered to the subject.
The invention further provides methods for modulating a T cell response comprising contacting a T cell with an agent which modulates a CD100 ligand-associated signal in the T cell. A T cell response is preferably T cell proliferation. The method also preferably includes contacting the T cell with a primary activation agent, such as an antibody to CD3 or at least one antigen.
Additional methods for modulating an interaction between immune cells and nerve cells in a subject by administering an amount of an agent which modulates a CD100 signal in the subject as well as methods for modulating and/or guiding axonal growth by contacting neurons with a modulating form of CD100, such that such that axonal growth is modulated and/or guided are also within the scope of the invention.